Hardening agent for affected tissues of the digestive system

ABSTRACT

The hardening agent contains a composition comprised of tannic acid and potassium aluminium sulfate in a ratio of tannic acid to potassium aluminium sulfate ranging from 10 to 1 to 1 to 50 and a stabilizing agent extracted from crude drugs of plants containing a phenol, flavon, flavonoid, catechin or a polycarboxylic acid.

This application is a continuation of Ser. No. 07/690,762 filed Apr. 24,1991, now U.S. Pat. No. 5,252,344.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a hardening agent for hardeningaffected tissues of the digestive system and, more particularly, to ahardening agent for hardening the affected tissues of the digestivesystem, suitable for curing the affected tissues thereof by withering,hardening, fixing or fibrillating the affected tissues thereof byinjecting a medicine or agent in a non-operative way into theesophagophleboma, piles, the site of the rectal prolapse, the site ofthe prolapse of the rectal mucosa and the affected uplift tissues(carcinomatous polyps) of the large intestine and the rectum underobservation by the proctoscope or endoscope or under X-ray examination.

2. Description of Related Art

Conventional hardening agents for curing the esophagophleboma and thepiles of a light or intermediate degree include, for example, ethanolamine of oleic acid, polydecanol, phenol almond oil and so on. The tumorof the digestive system is subjected to percutaneous ethanol injectiontherapy.

The piles of a severe degree, the rectal prolapse and the prolapse ofthe rectal mucosa are treated in an operative way.

Heretofore, as the method for non-operatively curing theesophagophleboma and the piles, there has been employed hardeningtherapy for hardening the affected tissues by injecting varioushardening agents into the affected tissues. Focuses of the affectedtissues are brought into an inactive state by withering, hardening,fixing or fibrillating the focuses thereof due to the embolism of theblood vessels of the affected tissues or due to the inflammation-inducedrepair reaction of the peripheral tissues. This hardening therapyrequires neither general anesthesia nor laparotomy. Hence, the risk isso small that this therapy presents the advantage that it canextensively be applied to weakening patients or old patients. Desirableconditions for a hardening agent include strong astringency andhemostatic effect. The aseptic inflammation reaction induced byinjection of the hardening agent can reduce the extent of edema,congestion and exudation if the acute inflammation reaction could beeliminated for a short period of time, thereby capable of preventing theaffected tissues from causing necrosis. When the aseptic inflammationreaction has been transferred to its chronic inflammation phase, thenthe tissues are withered, hardened, fixed and fibrillated and thefocuses disappear. Hence, the desired curing object can be achieved.

The conventional hardening agents, however, are limited in uses, as willbe described hereinafter. More specifically, such hardening agents ashaving strong action require injection under direct examination byX-rays while admixing a contrast medium with the hardening agent,because there is the risk of necrosing the tissues if they would beleaked from the blood vessels. On the other hand, hardening agentshaving mild action is so applied as to harden the affected tissues bywithering, hardening, fixation or fibrosis by injecting the tissues ofthe focal site outside the blood vessels with the hardening agents.Hence, in a state that the veins are varicose, the hardening agents arelittle employed solely. Particularly, for large esophagophleboma, atwo-step therapy is employed such that the hardening agent havingstronger action is injected directly into the vessels, while thehardening agent having milder action is injected into the surroundingtissues. Thus, the kinds of the hardening agents should be chosen inaccordance with the condition of a disease, and multi-injection requiredis laborious. Particularly, in curing the piles, the hardening agentsare so adapted as to be effective for a small amount of bleeding and alight degree of the prolapse of the anus, however, they are not soadapted as to be effective for such cases as requiring digitalreplacement of the anal prolapse caused during defecation or as beingslipping down from the anus and being ordinarily out of place. Thesecases require operative curing. Further, operative reparative fixationis applied to cases of the rectal prolapse and the prolapse of therectal mucosa, however, they are out of adaptation of the hardeningagents.

SUMMARY OF THE INVENTION

Therefore, the present invention has the object to provide a hardeningagent for hardening the affected tissues of the digestive organs soadapted as to be effectively applicable to the rectal prolapse, theprolapse of the rectal mucosa; the piles of a severe degree, and theaffected uplift tissues (carcinomatous polyps) of the large intestineand the rectum which could not so far be treated and cured by theconventional agents, while solving the disadvantages and problemsinherent in the conventional hardening agents and ensuring effectivenessand safety.

In other words, the present invention relates to an aqueous hardeningagent comprising a plurality of ingredients having action for theembolism of the blood vessels or action for hardening the tissues. Thehardening agent preferably has the pharmacological properties asfollows: (1) the aseptic acute inflammation reaction to be caused byinjection of the hardening agent is so light with respect to the extentof edema and congestion and so short with respect to the period of timeof inflammation due to the presence of an agent having action ofastringency; (2) its action of causing the embolism of the blood vesselsis accurate and appropriate and the tissues cause no necrosis even ifinjected outside the vessels; and (3) the fibrosis of the tissues can becompleted without leaving any stiffness in the course of repairing thechronic inflammation. The action of the tissues for repairing theinflammation can wither and inactivate the focuses. By the fixation ofthe tissues, the digestive organ is returned to its original position,without causing any risk of slipping down. The hardening agent accordingto the present invention is an aqueous injectable preparation so that itdoes not cause any side effect, such as the embolism of the vessels ofthe remote organs as reported for oleaginous, injectable preparations.

The present invention has another object to provide a hardening agentfor hardening the affected tissues of the digestive system, which isstable for a long period of time under room temperature and underconditions with light shaded.

In order to achieve the aforesaid objects, the present inventionconsists of a hardening agent for hardening affected tissues of thedigestive system, which comprises a composition containing tannic acidand potassium aluminum sulfate in a ratio of tannic acid to potassiumaluminium sulfate ranging from 10 to 1 to 1 to 50 and containing astabilizer comprising an extract from crude drags originating fromplants.

As a result of review on classical literature on Chinese traditionalmedicine, it is found that, back 2,000 years ago, publication titled"Emperor's Canon of Internal Medicine" describes to the extent that"piles are the result of blood vessels dilatation which may occurthrough abnormal defecation" and it has already implied that there is arelationship of occurrence of the piles with extension of the vesselsand abnormal defecation. Literature entitled "Plain Questions" setsforth the principle of a non-surgical therapy of "elevating the fallen"for the prolapse of the piles and the anal prolapse. Historical medicalbooks introduce various methods and preparations on the basis of thetheory of traditional Chinese medicine stating that "sour drugs may beused as astringent and punchery substance to control prolapse". Thebooks include, for example, "Compendium of Materia Medica", "Dan Xi XinFa" and "Tu Shu Ji Cheng". For instance, the book entitled "Dan Xi XinFa" states that "gallnut, alum, . . . treat piles to fumigate and towash by decoction".

It is to noted that the main ingredient of gallnut is tannic acid whichhas strong action of astringency against the tissues as well as actionof contracting the blood vessels and suppressing a variety ofmicroorganisms. It further has anti-exudation. And aluminium ion in asolution of potassium aluminium sulfate as an ingredient of white alumcauses inflammation on the local tissues and hardens and fibrillatesthem.

The hardening agent according to the present invention has the action ofhardening, fixing and fibrillating the site of the esophageal wall, therectal wall and the anal canal into which the agent has been injected,thereby hardening and curing the affected tissues of the digestiveorgans.

The hardening agent according to the present invention contains anadditive comprising an extract originating from crude drugs of plantssuch as, for example, Zingberis rhizoma, Mori folium, gallnut(Schinsandrae fructus), Plantaginis semen, Magnolicae cortex, Carthamiflos, Aurantii pericarpium, rosemary (Rosmarinus officinalis L.), sage(Salvia officinalis L.), thyme (Thymus vulgaris L.), marjoram (Origanusmajorana Moerch), oregano (Origanum vulgare L.), clove (Eugeniacaryopyllata Thumb), ginger (Zingiber officinale Roscoe), nutmeg(Myristica fragrans Houtt), mace (Myristica fragrans Houtt), turmeric(Curcuma longa L.), cinnamon (Cinnamomunzeylanicum blue), pepper (pipernigrum L.), and so on. The extract may preferably contain a phenol,flavon, flavonoid, catechin or polycarboxylic acid. The additive canstabilize the composition containing tannic acid and potassium aluminiumsulfate, and the ratio of tannic acid to potassium aluminium sulfateranges preferably from 10 to 1 to 1 to 50. The hardening agent accordingto the present invention in the form of an injectable preparation can bestored for a long period of time in a stable way.

Description will then be made of test procedures and results. The testsincludes acute toxicity test, inflammation-induced test on hind paws ofrats, cotton pellets granuloma test for rats, vessel endothelial cellinjury test, esophageal walls tissue injury-induced fibrillation testfor dogs, anorectal canal tissue injury-induced fibrillation test forrabbits, antibacterial test, carcinogenic tests for entericadministration of carcinogens to rats, the kind of preparations andstability test for preparations.

Acute Toxicity Test

The injectatable preparations of the hardening agents according to thepresent invention were intravenously or intraperitoneally administeredto groups of rats, each group consisting of 10 male mice of αα-typehaving weight of 20 grams plus or minus 2 grams. The results of deathwere observed in seven days after administration and the lethal dose 50(LD₅₀, ml/kg) was computed according to the Richfield-Wilcockson method.The test results are shown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Lethal Dose 50 (LD.sub.50, ml/kg)                                             Present Invention                                                                            i.v.       i.p.                                                ______________________________________                                        Preparation (1)                                                                              8.2 plus   22.5 plus                                                          or minus 0.4                                                                             or minus 0.9                                        Preparation (2)                                                                              7.6 plus   19.8 plus                                                          or minus 0.3                                                                             or minus 0.7                                        ______________________________________                                    

Inflammation-Induced Test on Hind Paws of Rats

1. Test Procedures

The hardening agents of the present invention, commercially availablehardening agent and physiological saline were administered to groups ofrats, each group consisting of 13 female and male rats having weight of220 grams plus or minus 20 grams and a variation in the volume of thehind paw (edema or swelling) was periodically measured. The rat's hindpaw on one side was administered with the hardening agent in the amountof 0.15 ml and with physiological saline in the amount of 0.15 ml as acontrol. The volumes of the hind paws were measured in 1 hours, 3 hours,5 hours, 12 hours, 24 hours, 72 hours, and 120 hours afteradministration and the acute inflammation reaction was considered usingan average increase value or rate of the hind paws causing inflammationas an indicator.

2. Test Results

The inflammation reaction was recognized in each group into which thehardening agent was administered. The extent of inflammation and days ofduration of inflammation were in the descending order of 5% phenolalmond oil, preparation (2) and preparation (1). The control into whichphysical saline was administered was light in the extent of inflammationand the inflammation induced in this control group was recovered withinshort. The test results are shown in Table 2 below.

                                      TABLE 2                                     __________________________________________________________________________    Inflammation-Induced Test on Hind Paws of Rats                                               Average Rate of Increasing Hind Paws                           Average        after Injection (ml)                                           Volume of Hind (edema rate, %)                                                Paws before    1    3    5    12   24   72   120                              Inflammation (ml)                                                                            hour hour hour hour hour hour hour                             __________________________________________________________________________    Prepara-                                                                            0.97     0.96**                                                                             1.08**                                                                             1.22**                                                                             0.92**                                                                             0.79**                                                                             0.25*                                                                              0.11                             tion (1)       98.9%                                                                              111.3%                                                                             125.7%                                                                             94.8%                                                                              81.4%                                                                              25.7%                                                                              11.3%                            Prepara-                                                                            1.01     1.10**                                                                             1.22**                                                                             1.35**                                                                             1.19**                                                                             0.96**                                                                             0.51*                                                                              0.30*                            tion (2)       108.9%                                                                             120.7%                                                                             133.6%                                                                             117.8%                                                                             95.0%                                                                              50.4%                                                                              29.7%                            5% phenol                                                                           0.98     1.15**                                                                             1.33**                                                                             1.48**                                                                             1.41**                                                                             1.30**                                                                             0.95**                                                                             0.78**                           almond oil     117.3%                                                                             135.7%                                                                             151.0%                                                                             143.8%                                                                             132.6%                                                                             96.9%                                                                              79.6%                            Physiolo-                                                                           1.00     0.145                                                                              0.162                                                                              0.26 0.18 0.06 --   --                               gical          14.5%                                                                              16.2%                                                                              26%  18%  6%                                         saline                                                                        __________________________________________________________________________     Notes:                                                                        *P < 0.01                                                                     **P < 0.001                                                              

Cotton Pellets Granuloma Tests for Rats

1. Test Procedures

The cotton pellets impregnated with the hardening agents of the presentinvention, commercially available hardening agent or physiologicalsaline were subcutatneously embedded on the back of the rat to therebyform granuloma. The granuloma induced by the cotton pellet was removedin seven days after administration and the weight of the granuloma wasmeasured. The tests were carried out by etherizing the rats andembedding aseptic cotton pellets (weighing 20 mg plus or minus 1 mg)impregnated with 0.1 ml of the hardening agent according to the presentinvention, 0.1 ml of the commercially available hardening agent or 0.1ml of physiological saline under the dermis on the back of the rat. Inseven days after embedding, the rats were killed to bleed by cuttingtheir heads and the granuloma induced by the cotton pellets was removedand weighed by electronic balance.

2. Test Results

The weight of the cotton pellets granuloma was in the descending orderof the preparation (1), the preparation (2), 5% phenol almond oil andphysiological saline. The test results are shown in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        Cotton Pellets Granuloma Tests for Rats                                       Test       No. of  Weight of Cotton                                                                             t-Accep-                                    Agents     Rats    Pellets Granuloma                                                                            tance                                       ______________________________________                                        Preparation (1)                                                                          10      485 mg plus or P < 0.001                                                      minus 55.12 mg                                             Preparation (2)                                                                          10      430 mg plus or P < 0.001                                                      minus 43.38 mg                                             5% Phenol  10      297 mg plus or P < 0.01                                    almond oil         minus 50.23 mg                                             Physiological                                                                            10      176 mg plus or --                                          saline             minus 12.28 mg                                             ______________________________________                                    

Vessel Endothelial Cell Injury Tests

1. Procedures for Preparation of Samples

The vessel endothelial cells were obtained by treating the umbilicalveins of the newborn with trypsin in conventional manner and subculturedin RITC80-7 culture medium. The resulting cell suspension was employedfor tests. The materials to be tested includes the preparation (1) ofthe hardening agent of the present invention, the preaparation (2)thereof, and a 5% ethanol amine oleate (EO) solution. The test materialswere employed as 2-fold, 5-fold, 10-fold, 20-fold and 100-fold dilution.

2. Test Procedures

A mixture of 50 microliters of the hardening agent of the aforesaidconcentration with 50 microliters of the aforesaid cell suspension wasmixed with 100 microliters of a 0.2% trypan blue dyeing solution.Immediately after mixing, the resulting mixture was transferred to ahemacytometer and the number of pigment-dyed dead cells was computed,thereby calculating a viability rate of cells. A control group in whichonly the culture medium was added to the vessel endothelial cellsuspension gave a viability rate of cells as high as 80%.

The extent of injury of cells induced by addition of the hardening agentwas rated from severe (+++), intermediate (++), low (+), and very low(-). The severe rating (+++) of injury means that the viability rate ofthe cells ranges from 0% to 20%; the intermediate rating (++) of injurymeans the viability rate of the cells ranging from 20% to 50%; the lowrating (+) of injury means the viability rate of the cells ranging from50% to 80%; and the very low rating (-) of injury means the viabilityrate of the cells ranging from 80% or higher. As diluting solutions wereemployed physiological saline and serum.

3. Results

The hardening agents diluted to a low extent caused injury in the vesselendothelial cells. It is further noted that a lower concentration of thehardening agent changes the extent of injury from severe rating to lowrating. The results may vary with the kind of the hardening agent. Itcan be noted, however, that dilution with serum has reduced the actioncaused by injury. Further, the extent of injury of the vesselendothelial cells corresponds to an initial stage of the vessel embolismaction of the affected tissues. The test results are shown in Table 4below.

                  TABLE 4                                                         ______________________________________                                        Injury of Vessel Endothelial Cells of Rats                                    by Hardening Agents                                                                       Dilutions of Hardening Agents                                     Hardening                                                                              Diluting 2-      5-    10-   20-  100-                               Agent    Solution fold    fold  fold  fold fold                               ______________________________________                                        Prepara- Physiolo-                                                                              +++     +++   +++   -    -                                  tion (1) cal saline                                                           Prepara- Serum    +++     ++    +     -    -                                  tion (1)                                                                      Prepara- Physiolo-                                                                              +++     +++   +++   -    -                                  tion (2) cal saline                                                           Prepara- Serum    +++     ++    +     -    -                                  tion (2)                                                                      5% EO    Physiolo-                                                                              +++     +++   +++   +    -                                           cal saline                                                           5% EO    Serum    +++     +++   +++   -    -                                  ______________________________________                                    

Esophageal Wall Tissues Injury-Induced Fibrillation Tests for Dogs

1. Method of Operation

Fifteen mixed-breed dogs having weight ranging from 8 kg to 12 kg weregrouped into three groups. The dogs went on a fast for a day beforeoperation and intravenously administered dropwise with 500 ml of aRinger's mixture with 1 gram of cefamezine, and they were intravenouslyanaesthetized with pentobarbital sodium at the rate of 20 mg perkilogram of the body weight. After anaesthesia, the pleura mediastinumwas cut open to expose and incise the lower esophagus lengthwise andadministered with the preparation (1) and the preparation (2) of thehardening agent according to the present invention and a 5% phenolalmond oil (briefly called Pao hereinafter) solution in the quantity of1 ml through a 27G two-step injectable needle under direct examination.After the bleeding has stopped completely, the pleura mediastinum cutopen was seamed with silky thread while inserting a drain. After the airwas removed, the drain was removed.

2. Test Procedures

After injection of the testing hardening agents into three groups ofdogs, the dogs were killed periodically, i.e. day 1, 3, 7, 14 and 28,and the esophagus was taken out. The esophagus taken out was visuallyobserved for states of the mucosa and serofluid of the site into whichthe agent has been injected, in terms of edema, necrosis and ulceration,and the results are rated from severe (+++), intermediate (++), light(+) and low (-).

After visual observation, the esophagus taken out was sliced in a widthas small as 3 mm and fixed by a 10% formalin solution. Thereafter, thesliced tissues were further sliced to a thickness of 3 microns by amicrotome and then dyed with hematoxylin-eosine for microscopicobservation for cytoinfiltration, fibrocyte, fibrosis and an extent ofcuring injury or damages of tissues of the newborn vessels.

3. Test Results

Acute inflammation such as edema and cytoinfiltration was observed ineach of the groups in which the agent was administered. The extent anddays of duration in this case were in the descending order of the 5%phenol almond oil solution, the preparation (2) and the preparation (1). The extent of chronic inflammation reaction such as fibrocyte andfibrosis was in the descending order of the preparation (1) , thepreparation (2) and the 5% phenol almond oil solution. The results areshown in Table 5 below.

                                      TABLE 5                                     __________________________________________________________________________    Visual and Histological Observations                                          Day after                                                                           Hardening                                                                           Necro-  Cytoinfil-                                                                         Ulcera-                                                                            Newborn                                                                            Fibro-                                                                            Fibril-                                Operation                                                                           Agents                                                                              sis Edema                                                                             tration                                                                            tion Vessels                                                                            blast                                                                             lation                                 __________________________________________________________________________    Day 1 Prep. (1)                                                                           -   ++  ++   -    -    -   -                                            Prep. (2)                                                                           -   ++  ++   -    -    -   -                                            Pao   ++  +++ ++   -    -    -   -                                      Day 3 Prep. (1)                                                                           -   -   +    -    -    +   -                                            Prep. (2)                                                                           -   +   +    -    -    +   -                                            Pao   ++  +++ +++  ++   -    -   -                                      Day 7 Prep. (1)                                                                           -   -   -    -    ++   +++ ++                                           Prep. (2)                                                                           -   -   -    -    ++   + ++                                                                              ++                                           Pao   +   -   +    +    +    +   -                                      Day 14                                                                              Prep. (1)                                                                           -   -   -    -    +++  +++ +++                                          Prep. (2)                                                                           -   -   -    -    +++  +++ +++                                          Pao   -   -   -    -    ++   ++  ++                                     Day 28                                                                              Prep. (1)                                                                           -   -   -    -    +++  +++ +++                                          Prep. (2)                                                                           -   -   -    -    +++  +++ +++                                          Pao   -   -   -    -    +++  +++ +++                                    __________________________________________________________________________

Anorecral Canal Tissue Injury-Induced Fibrillation Tests

1. Method of Operation

Fifteen domestic rabbits of white type having weight from 2.8 kg to 3.2kg were equally grouped into three groups. The rabbits went on a fastfor a day before operation and an enema of 10 ml of glycerin solutionwas applied on the day of operation. The rabbits were intravenouslyadministered dropwise with 100 ml of a 5% glucose solution containing0.5 gram of cefamezine, and they were anaesthetized by intravenousadministration of pentobarbital sodium at the rate of 25 mg per kilogramof the body weight. After anaesthesia, the peritoneum was cut open byceliotomy to expose and incise the rectum and anal canal lengthwise andthe preparation (1) or the preparation (2) of the hardening agent of thepresent invention or a 5% phenol almond oil solution (briefly called Paohereinafter) was injected in the quantity of 0.3 ml through anorder-made 29G two-step injectable needle under direct examination.After the bleeding has been stopped, the lengthwise incised rectal andanal canal portions were sutured with silky thread while inserting adrain and closing the stomach. The drain was removed after dischargingthe solution from stomach.

2. Test Procedures

After injection of the testing hardening agents into three groups of therabbits, the rabbits were killed periodically, i.e. day 1, 3, 5, 7 and14, and the rectal and anal canal were taken out. The organ sectionstaken out was visually observed for states of the mucosa and serofluidof the site into which the agent has been injected, in terms of edema,necrosis and ulceration, and the results are rated from severe (+++),intermediate (++), light (+) and low (-).

After visual observation, the organ sections taken out were sliced in awidth as small as 3 mm and fixed by a 10% formalin solution. Thereafterthe sliced tissues were sliced to a thickness of 3 microns by amicrotome and then dyed with hematoxylin-eosine for microscopicobservation for cytoinfiltration, fibrocyte, fibrosis and an extent ofcuring injury or damages of tissues of the newborn vessels.

3. Test Results

Acute inflammation such as edema and cytoinfiltration was observed ineach of the groups in which the agent was administered. The extent anddays of duration in this case were in the descending order of the 5%phenol almond oil solution, the preparation (2) and the preparation (1).The extent of chronic inflammation reaction such as fibrocyte andfibrosis was in the descending order of the preparation (1), thepreparation (2) and the 5% phenol almond oil solution. The results areshown in Table 6 below.

                                      TABLE 6                                     __________________________________________________________________________    Day after                                                                           Hardening                                                                           Necro-  Cytoinfil-                                                                         Ulcera-                                                                            Newborn                                                                            Fibro-                                                                            Fibril-                                Operation                                                                           Agents                                                                              sis Edema                                                                             tration                                                                            tion Vessels                                                                            blast                                                                             lation                                 __________________________________________________________________________    Day 1 Prep. (1)                                                                           -   ++  ++   -    -    -   -                                            Prep. (2)                                                                           -   ++  ++   -    -    -   -                                            Pao   ++  +++ ++   ++   -    -   -                                      Day 3 Prep. (1)                                                                           -   -   +    -    -    ++  -                                            Prep. (2)                                                                           +   +   +    -    -    ++  -                                            Pao   ++  ++  +++  ++   -    -   -                                      Day 5 Prep. (1)                                                                           -   -   -    -    ++   +++ +++                                          Prep. (2)                                                                           -   -   -    -    ++   ++  + ++                                         Pao   +   -   +    +    +    +   -                                      Day 7 Prep. (1)                                                                           -   -   -    -    +++  +++ +++                                          Prep. (2)                                                                           -   -   -    -    +++  +++ +++                                          Pao   +   -   -    -    +    +   +                                      Day 14                                                                              Prep. (1)                                                                           -   -   -    -    +++  ++  +++                                          Prep. (2)                                                                           -   -   -    -    +++  ++  +++                                          Pao   -   -   -    -    +++  +++ ++                                     __________________________________________________________________________

Antibacterial Tests

The preparation (1) of the hardening agent according to the presentinvention was tested for its antibacterial activity by plate method andtest tube method in conventional manner. The solid cuture media employedfor plate method were prepared by adding a mixture of given amounts ofL-cystein, agar, sodium chloride, glucose, yeast extract and caseinpeptone with 50 ml of the preparation (1) to 920 ml of water, dissolvingthem in a warm bath and pouring a given amount of the culture medium indishes which in turn were sterilized in an autoclave at 121° C. for 20hours and employed for tests followed by the return to room temperature.On the other hand, the liquid culture media to be employed for the testtube method were prepared by using the same mixture, except for agar,and pouring the media into test tubes with cotton lids. The test tubeswere employed in the same manner after sterilization in conventionalmanner. The incubation was carried out in both cases in an incubator at37° C. for 3 days.

The antibacterial activity of the preparation (1) was determined asactive when a colony or colonies was or were visually observed on theincubated plate culture media, while as inactive when no colory wasvisually observed thereon. On the other hand, for the test tube method,the preparation (1) was determined as active when the culture medium wastransparent, while as inactive when the culture medium was turbid. InTable 7, the minus sign means active and the plus sign means inactive.

                  TABLE 7                                                         ______________________________________                                        Antibacterial Tests                                                                       Stock Strains                                                              Dilutions       B.         Ps.                                       Method of                                                                              of Prepa-                                                                              E.     sub- Staph.                                                                              aeru- Clostri-                            Tests    ration (1)                                                                             coli   tilis                                                                              aureus                                                                              ginosa                                                                              dium sp.                            ______________________________________                                        Plate    stock    -      -    -     -     -                                            soln                                                                 Plate    1:3      -      -    -     -     -                                   Plate    1:10     -      -    -     -     -                                   Plate    1:30     +      -    -     -     -                                   Test Tube                                                                              stock    -      -    -     -     -                                            soln                                                                 Test Tube                                                                              1:3      -      -    -     -     -                                   Test Tube                                                                              1:10     -      -    -     -     -                                   Test Tube                                                                              1:30     +      -    +     +     +                                   ______________________________________                                    

Carcinogenic Tests by Enteric Administration of Carcinogen to Rats

The enteric administration of carcinogens to rats may cause tumors onthe distocolon and the rectum. It is to be noted herein that the groupof rats in which feed containing a large amount of animal fats and oilhas been fed for a long period of time particularly after administrationof the carcinogens may cause tumors at a higher probability than thegroup in which feed containing a standard amount of animal fats has beenfed. It has been found that the administration of the hardening agentsaccording to the present invention to the affected tissues of the ratsunder endoscopic observation can suspend propagation of the affecteduplift tissues. Thus, this method of curing can prolong the life of therats. Test procedures and test results will be described hereinafter.

1. Test Procedures

Fifty male rats of F344 type weighing from 120 grams plus or minus 20grams were grouped into four groups, two out of the four groups, eachconsisting of twenty male rats and two ouf of the four groups,consisting of five rats. The rats of the two groups, each consisting oftwenty rats, and of the one out of the two groups, each consisting offive rats, were administered with a carcinogen asN-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by injecting a 0.25% aqueoussolution of MNNG to the depth of 3 cm from the anus using a polyethylenetube at the rate of 0.3 ml per once a day consecutively for four hoursafter anaesthesia to a light degree with ether prior to entericadministration. A control group of rats consisting of five rats waslikewise administered with physiological saline. The standard-fat feedcontained swine fats at the rate of 20%, while the high-fat feedcontained fats at the rate of 5%. The rats in the three groups were fedwith the standard-fat feed during a period of time for which MNNG hasbeen administered, and the group consisting of twenty rats treated withthe hardening agent of the present invention as preparation (5) was fedwith the high-fat feed after the four week duration of administrationwith MNNG, while the non-treated group consisting of twenty rats was fedwith the high-fat feed and the non-treated group consisting of five ratswas further fed with the standard-fat feed. The control group of fiverats which had been treated with physiological saline was fedcontinuously with the standard-fat feed over the entire period of testtime. The rats with the preparation (5) of the present inventionadministered were periodically examined for distocolon and rectum by anendoscope. When the affected uplift tissues of the digestive organ ofthe rats were recognized, then the preparation (5) of the hardeningagent of the present invention was locally injected in the amount of0.05 ml to 0.5 ml into the affected tissues thereof and the tissuessurrounding the affected tissues. The duration for feeding the ratslasted 18 months after administration of the MNNG or the physiologicalsaline, and all the rats alive after the duration of feeding were killedto observe the presence or absence of tumors on the distocolon and therectum.

2. Test results

It can be noted that the number of the alive rats of the rats in thethree groups to which MNNG had been administered was decreasing as theduration of feeding elapses. For the two groups in which the rats werefed with the high-fat feed, cases such as malappetite, reduction in bodyweight, bloody excrement, etc. were recognized and the rats have causedthe anal prolapse, obstruction of the intestines and intussusception. Itcan be noted that, for the non-treated groups, the number of the aliverats has decreased to a statistically significant extent, as comparedwith the groups treated with the preparation (5) of the presentinvention. The number of the alive rats in the three groups was found tobe twenty-one rats after of a lapse of eighteen months of feeding and itwas found that the number of tumors per one rat ranged from five totwelve and that an average per one rat was 8.2 tumors. It can further benoted that the rats in the groups treated with the preparation (5) ofthe present invention has caused fibrillation of the affected tissuesand granulation, however, no newborn tumors were recognized. The testresults are shown in Table 8 below.

                                      TABLE 8                                     __________________________________________________________________________    Carcinogenic Tests by Enteric Administration of Carcinogen to Rats            Duration of  Kind of Feed                                                                            Months of Feeding after                                Administration of                                                                          after Administration                                                                    Administration of MNNG or                              MNNG or Physiological                                                                      of MNNG or                                                                              Physiological Saline                                   Saline (4 weeks;                                                                           Physiological                                                                           & (No. of Alive Mice)                                                                       t-Accep-                                 Standard-Fat Feed)                                                                         Saline    0   6  12  18 tance                                    __________________________________________________________________________    Group of                                                                      Administration of MNNG                                                        Non-Treated Group                                                                          Standard   (5)                                                                               (5)                                                                              (4)                                                                               (3)                                                                             -                                        Group of Prep. (5)                                                                         High-Fat  (20)                                                                              (19)                                                                             (17)                                                                              (15)                                                                             <0.01                                    Non-Treated Group                                                                          High-Fat  (20)                                                                              (18)                                                                             (10)                                                                               (3)                                                                             <0.01                                    Control Group of                                                                           Standard   (5)                                                                               (5)                                                                              (4)                                                                               (4)                                                                             -                                        Administration of                                                             Physiological Saline                                                          __________________________________________________________________________

Description will be made of the preparations for the hardening agentsaccording to the present invention.

PREPARATION (1)

    ______________________________________                                        Tannic acid from     0.15    gram                                             Schinsandrae fructus                                                          Potassium aluminium  4.00    grams                                            sulfate                                                                       Sodium citrate       1.50    grams                                            Dextran 40           1.00    gram                                             Glycerin             10.00   grams                                            Extract from crude drugs                                                                           4.00    ml                                               of various plants* or**                                                       Sodium hydrogen sulfite                                                                            0.10    gram                                             Disodium calcium     0.01    gram                                             edetoate                                                                      Distilled water      to make 100 ml                                           (adjusted to pH 4-5 with a pharmacologically                                  intoxic acid or alkali)                                                       ______________________________________                                         Notes:                                                                        *The various plants contain Zingberis rhizoma, Mori folium, Schinsandrae      fructus, Plantaginis semen, Magnolicae cortex, Carthami flos and Aurantii     pericarpium and they were extracted with 2 ml of an ethanol aqueous           solution.                                                                     **The various plants include rosemary, sage, thyme, marjoram, oregano,        clove, ginger, nutmeg, mace, turmeric, cinnamon and pepper, and the plant     were extracted with 2 ml of an ethanol aqueous solution and ampouled in       the amount of 10 ml into colorless hardglass amboules and sterilized unde     highpressurized steam in conventional way. The gases contained in the         ampoules were replenished with nitrogen gas.                             

PREPARATION (2)

    ______________________________________                                        Tannic acid from     0.20    gram                                             Schinsandrae fructus                                                          Potassium aluminium  2.00    grams                                            sulfate                                                                       Sodium citrate       1.50    grams                                            Dextran 40           1.00    gram                                             Glycerin             10.00   ml                                               Sodium hydrogen sulfite                                                                            0.10    gram                                             Disodium calcium     0.01    gram                                             edetoate                                                                      Distilled water      to make 100 ml                                           (adjusted to pH 4-5 with a pharmacologically                                  intoxic acid or alkali)                                                       ______________________________________                                    

The solution in the amount of 10 ml was poured into colorless glassampoules which in turn were sterilized with high-pressurized steam inconventional manner and which were replenished with nitrogen gas.

PREPARATION (3)

This preparation (3) was employed by dissolving the preparations A and Bon place.

Preparation A

    ______________________________________                                        Tannic acid from      0.015   gram                                            Schinsandrae fructus                                                          Extract from crude drugs                                                                            0.20    ml                                              of various plants*                                                            Sodium hydrogen sulfite                                                                             0.01    gram                                            Distilled water       10.00   ml                                              (adjusted to pH 4-5 with a pharmacologically                                  intoxic acid or alkali)                                                       ______________________________________                                         Notes:                                                                        *The various plants contain Zingberis rhizoma, Mori folium, Schinsandrae      fructus, Plantaginis semen, Magnolicae cortex, Carthami flos and Aurantii     pericarpium and the crude drugs were extracted with an ethanol aqueous        solution.                                                                

A solution of the preparation A was filtered by suction using a0.22-micron membrane filter and the filtrate was poured into ampoules orvials which in turn were cooled and frozen well to allow ice to sublimeunder high vacuum condition and the resulting preparation to dry.

Preparation B

    ______________________________________                                        Potassium aluminium sulfate                                                                         4.00    grams                                           Sodium citrate        1.50    grams                                           Dextran 40            1.00    gram                                            Glycerin              10.00   ml                                              Disodium calcium edetoate                                                                           0.01    gram                                            (adjusted to pH 4-5 with a pharmacologically                                  intoxic acid or alkali)                                                       ______________________________________                                    

The colorless hard glass ampoules were poured at the rate of 10 ml andsterilized under high-pressurized steam.

PREPARATION (4)

The preparation (4) was prepared by dissolving it on site.

    ______________________________________                                        Tannic acid from      0.20    gram                                            Schinsandraw fructus                                                          Potassium aluminium   4.00    grams                                           sulfate                                                                       Sodium citrate        1.00    grams                                           Dextran 40            1.00    gram                                            Extract from crude drugs                                                                            4.00    ml                                              of various plants*                                                            Sodium hydrogen sulfite                                                                             0.05    gram                                            Disodium calcium      0.01    gram                                            edetoate                                                                      Distilled water       to make 100 ml                                          (adjusted to pH 4-5 with a pharmacologically                                  intoxic acid or alkali)                                                       ______________________________________                                         Notes:                                                                        *The various plants contain Zingberis rhizoma, Mori folium, Schinsandrae      fructus, Plantaginis semen, Magnolicae cortex, Carthami flos and Aurantii     pericarpium and extracted with an ethanol aqueous solution.              

A solution of the preparation (4) was filtered by suction using a0.22-micron membrane filter and the filtrate was poured in the amount of10 ml into ampoules or vials which in turn were cooled and frozen wellto allow ice to sublime under high vacuum condition and the preparationto dry.

PREPARATION (5)

    ______________________________________                                        Tannic acid           4.00    grams                                           Potassium aluminium   4.00    grams                                           sulfate                                                                       Sodium citrate        0.30    gram                                            Dextran 40            2.00    gram                                            Conc. glycerin        2.50    ml                                              Chlorobutanol         0.30    gram                                            Extract from crude drugs                                                                            2.00    grams                                           of various plants                                                             Sodium hydrogen sulfite                                                                             0.10    gram                                            Sodium edetoate       0.01    gram                                            Distilled water       to make 100 ml                                          (adjusted to pH 4-5 with a pharmacologically                                  intoxic acid or alkali)                                                       ______________________________________                                         Notes:                                                                        *The various plants contain orange skin, orange and lemon were extracted      with 4 ml of an ethanol aqueous solution.                                

The preparation was poured into 50-ml colorless glass vials andsterilized with under high pressures in conventional manner. The vialswere replenished with nitrogen gas.

Stability Tests for Preparations

Colorless hard glass ampoules were filled with 10 ml of a composition ofthe hardening agents according to the present invention. After theampoules were replenished with nitrogen gas, they were sterilized at121° C. for 20 minutes by steam under high pressures in conventionalmanner. The hardening agents according to the present invention wereaquous after sterilization and the colors are colorless or pale yellow.

Under various storage conditions, the preparations (1) and (2) aretested for stability for a long period of time. The preparation (1) wasstable for three years and three months under conditions under whichlight scattered in a room. The preparation (1) turned yellow in arelatively short period of time under exposure to light (Xenon lamp)under room temperature. On the other hand, the preparation (2) was foundstable for six months under light scattering conditions in a room andturned yellow in a short period of time under conditions under whichXenon lamp has been exposed at room temperature. The test results areshown in Tables 9 and 10.

                  TABLE 9                                                         ______________________________________                                        Tests for Stabilizing Preparations                                            under Light Scattering Conditions in Room                                     ______________________________________                                        Storage conditions:                                                                          Light scattering conditions in                                                room at room temperature (white                                               light, 100 lux)                                                Storage vessel:                                                                              Colorless hard glass ampoules                                                 (five ampoules in a paper box)                                 Preparations:  Preparation (1) and preparation (2)                            Storage Duration                                                              (months)       Preparation (1)                                                                           Preparation (2)                                    ______________________________________                                          0            Colorless-  Colorless-                                                        pale yellow pale yellow                                          1            Colorless-  Colorless-                                                        pale yellow pale yellow                                          2            Colorless-  Colorless-                                                        pale yellow pale yellow                                          3            Colorless-  Colorless-                                                        pale yellow pale yellow                                          6            Colorless-  Colorless-                                                        pale yellow pale yellow                                         12            Colorless-  yellow                                                            pale yellow                                                     18            Colorless-  yellow                                                            pale yellow                                                     24            Colorless-  yellow                                                            pale yellow                                                     30            Colorless-  yellow-                                                           pale yellow brown                                               36            Colorless-  yellow-                                                           pale yellow brown                                               39            Colorless-  yellow-                                                           pale yellow brown                                              ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        Tests for Stabilizing Preparations                                            under Light Exposing Conditions in Room                                       ______________________________________                                        Storage conditions:                                                                          Light exposing conditions in                                                  room at room temperature (Xenon                                               light)                                                         Storage vessel:                                                                              Colorless hard glass ampoules                                  Preparations:  Preparation (1) and preparation (2)                            Storage Duration                                                              (months)       Preparation (1)                                                                           Preparation (2)                                    ______________________________________                                          0            Colorless-  Colorless-                                                        pale yellow pale yellow                                          1            Colorless-  Colorless-                                                        pale yellow pale yellow                                          2            Colorless-  Colorless-                                                        pale yellow pale yellow                                          3            Colorless-  yellow                                                            pale yellow yellow                                              10            Colorless-  Colorless-                                                        pale yellow pale yellow                                         14            yellow      yellow-                                                                       brown                                               24            yellow      yellow-                                                                       brown                                              ______________________________________                                    

The hardening agents according to the present invention show hardeningactivity of the affected tissues of the digestive system. Further, thepresent invention can provide the novel hardening agents for curing theesophagophleboma, piles and affected uplift tissues (carcinomatouspolyps).

The hardening agents according to the present invention can demonstratestability for a long period of time under shaded conditions and secureexcellent quality.

What is claimed is:
 1. An injectable preparation for hardening thetissue of digestive organs comprising a tissue-hardening effectiveamount of tannic acid and potassium aluminium sulfate as well as astabilizing agent extracted from crude drugs of plants selected from thegroup consisting of Zingberis rhizoma, Mori folium, Schinsandrae cortex,Carthami sage, thyme, marjoram, oregano, clove, ginger, nutmeg, mace,turmeric, cinnamon, pepper and combinations thereof.
 2. The injectablepreparation as claimed in claim 1, wherein the stabilizing agent is anextract of aromatic crude drugs of the plants containing essential oilor an extract from the crude drugs of the plants containing organicacids.
 3. The injectable preparation as claimed in claim 1, wherein thestabilizing agent contains phenol, flavon, flavonoid, a catechin or apolycarboxylic acid.
 4. The injectable preparation as claimed in claim1, wherein the ratio of tannic acid to potassium aluminium sulfateranges from 10 to 1 to 1 to 50.